Quercus acuta is an evergreen broadleaf tree that grows in the warm-temperate regions of Korea and Japan. Its habitats and populations are being destroyed, and a new northernmost limit of distribution has now been reported. To further our scientific understanding of its conservation and phylogeography, we isolated and characterized 13 microsatellite loci. An analysis of diversity was conducted among 35 individuals on Hong-do Island of Jeollanam-do, South Korea. Variability of the markers was also tested for 11 individuals from Jeju-do. At the population level, alleles numbered 2 to 12 and the observed and expected heterozygosities ranged from 0.0909 to 0.9143 and from 0.0909 to 0.9364, respectively. Those 13 loci were also tested for cross-species amplification in three other evergreen Quercus species within the same subgenus Cyclobalanopsis. In all, 6 of 13 loci could be amplified for all three species. The microsatellite markers described here provide a powerful genetics tool for population, conservation, systematics, and phylogeographic studies, not only for Q. acuta but also for other evergreen Quercus species.